Immune regulation is a fundamental property of the immune system capable of controlling immune mediated events which may result in significant disease. Cellular populations capable of immune regulation have been defined which include CD4+CD25 + Treg cells, and alpha/betaTCR+ CD4+NK1.1+ NK-T cells. A major setting in which immunological reactions play both a potentially positive and negative role is following hematopoietic cell transplantation where graft vs tumor (GVT) reactions are capable of controlling malignancy whereas, graft vs host disease (GVHD) is a major cause of morbidity and mortality. We have recently discovered that CD4+CD25+ regulatory T cells are capable of suppressing GVHD without interfering with GVT reactions. We have developed novel methodologies to probe the biology and mechanisms underlying this observation based upon the use of bioluminescent imaging (BLI). By introducing the luciferase gene into cell populations of interest the trafficking and survival of both tumor and T cell populations can be readily assessed. We have demonstrated that BLI is capable of detecting as few as 100-1,000 cells in vivo in a non-invasive and quantitative manner. In this Project we will utilize these techniques as well as recently derived transgenic animals which constitutively express the luc gene for the study the role of regulatory T cells in complex biological events such as GVHD and GVT which require in vivo analysis in the context of the appropriate tissue environment and blood flow. We will also explore the biological activity of ex vivo expanded regulatory T cells. With the use of bifunctional reporter gene constructs combinining the power of immunofluorescence based cell sorting and tissue analysis with gfp to that of BLI-based evaluation of in vitro and in vivo function with luciferase we will evaluate the mechanisms underlying the suppression of GVHD and preservation of GVT by CD4+CD25+ regulatory T cells. We will also utilize animals deficient in key effector molecules to probe the mechanisms underlying these biological observations. Additional studies will be performed to compare and contrast the biological activity of CD4+CD25+ Treg cells with alpha/betaTCR +CD4 +NK1.1 + cells. These studies have important implications in the evaluation of complex biological events such as GVHD and GVT and also are critical to the potential clinical application of this novel cellular population of regulatory T cells.